Potential anti-bacterial drug target: Structural characterization of 3,4-dihydroxy-2-butanone-4-phosphate synthase from Salmonella typhimurium LT2
Potential anti-bacterial drug target: Structural characterization of 3,4-dihydroxy-2-butanone-4-phosphate synthase from Salmonella typhimurium LT2
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DOI：
10.1002/prot.22837
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发表时间：
2010-12-01
影响因子：
2.9
通讯作者：
Karthikeyan, Subramanian
中科院分区：
文献类型：
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作者：
Kumar, Pankaj;Singh, Mirage;Karthikeyan, Subramanian
ribB基因编码的3，4-二羟基-2-丁酮-4-磷酸合酶（DHBPS）是核黄素生物合成途径中的第一个酶，催化5-磷酸核酮糖（Ru 5 P）转化为3，4-二羟基-2-丁酮-4-磷酸和甲酸。DHBPS是开发抗菌药物的一个有吸引力的目标，因为这种酶对病原体是必需的，但在人类中不存在。沙门氏菌的重组DHBPS酶需要镁离子才能发挥其活性，并在37 ℃下以199 nmol min（-1）mg（-1）的速率催化Ru 5 P形成3，4-二羟基-2-丁酮-4-磷酸，K-m值为116 μ M。此外，我们已经确定了沙门氏菌DHBPS与硫酸根，Ru 5 P和硫酸根-锌离子复合物的晶体结构，分辨率分别为2.80，2.52和1.86埃。这些晶体结构的分析表明，负责酸碱催化的酸性环（残基34-39）在活性位点不存在底物或金属离子时是无序的。在结合底物或硫酸根和金属离子后，酸性环变得稳定，采用闭合构象并与底物相互作用。我们的结构第一次表明，底物Ru 5 P单独的结合是足够的稳定的酸性活性位点环到一个封闭的构象。此外，Glu 38残基从酸性活性位点环后Ru 5 P结合，这有助于定位第二个金属离子，稳定Ru 5 P和反应中间体的构象变化。这是DHBPS与任何真细菌的底物或金属离子复合的第一个结构报告。
3,4-Dihydroxy-2-butanone-4-phosphate synthase (DHBPS) encoded by ribB gene is one of the first enzymes in riboflavin biosynthesis pathway and catalyzes the conversion of ribulose-5-phosphate (Ru5P) to 3,4-dihydroxy-2-butanone-4-phosphate and formate. DHBPS is an attractive target for developing anti-bacterial drugs as this enzyme is essential for pathogens, but absent in humans. The recombinant DHBPS enzyme of Salmonella requires magnesium ion for its activity and catalyzes the formation of 3,4-dihydroxy-2-butanone-4-phosphate from Ru5P at a rate of 199 nmol min(-1) mg(-1) with K-m value of 116 mu M at 37 degrees C. Further, we have determined the crystal structures of Salmonella DHBPS in complex with sulfate, Ru5P and sulfate-zinc ion at a resolution of 2.80, 2.52, and 1.86 angstrom, respectively. Analysis of these crystal structures reveals that the acidic loop (residues 34-39) responsible for the acid-base catalysis is disordered in the absence of substrate or metal ion at the active site. Upon binding either substrate or sulfate and metal ions, the acidic loop becomes stabilized, adopts a closed conformation and interacts with the substrate. Our structure for the first time reveals that binding of substrate Ru5P alone is sufficient for the stabilization of the acidic active site loop into a closed conformation. In addition, the Glu38 residue from the acidic active site loop undergoes a conformational change upon Ru5P binding, which helps in positioning the second metal ion that stabilizes the Ru5P and the reaction intermediates. This is the first structural report of DHBPS in complex with either substrate or metal ion from any eubacteria.